Sustained release pharmaceutical compositions for the parenteral administration of hydrophilic compounds

ABSTRACT

Stable, biologically compatible pharmaceutical compositions in the form of water-in-oil microemulsions (w/o), for the sustained release by parenteral administration of active ingredients which are hydrophilic or are made hydrophilic by suitable derivatization, a process for the preparation of said microemulsions and the use thereof.

FIELD OF THE INVENTION

The present invention relates to sustained release pharmaceuticalcompositions for the parenteral administration of active ingredientswhich are hydrophilic or are made hydrophilic by suitablederivatization, in the form of stable, biologically compatible andeasily preparable water-in-oil microemulsions (w/o). More particularly,peptide active ingredients or biologically active oligo- orpolysaccharides, for which protection from the immediate attack by thehydrolytic enzymes present in living organisms as well as sustainedrelease in time, to avoid repeated administrations, are desirable, areadvantageously formulated through said microemulsions. Said formulationsfor the therapeutical use can be injected without problems orsignificant side effects and are easily prepared industrially, providinga remarkable technical improvement.

PRIOR ART

Microemulsions can be generally defined as optically isotropic systems,not birifrangent under polarized light observation, transparent,thermodynamically stable, of extremely reduced size, with dropletdiameter ranging from 5 to 200 nm, produced by dispersion of twoimmiscible liquids which are stabilized by the presence of emulsifiers,which modify the chemical-physical properties of the separation surfacebetween the two liquids, reducing substantially to zero the interfacialtension. An oil, water, a surfactant or tenside and optionally of aco-surfactant o co-tenside should usually be present for microemulsionsto form; the tendency to form a water-in-oil (w/o) or oil-in-water (o/w)microemulsion depends on the mutual proportions of aqueous and oilyphases as well as on the nature of the surfactant. In particular,ternary phase diagrams having as components water, a hydrophobiccompound and a mixture of surfactant and co-surfactant, obtainedaccording to experimental data, allow to single out the area in whichw/o and o/w microemulsions exist and are stable. For example, Aboofazeliet al (Int.J.Pharm. 111 (1994) 63–72) studied the capability of variouscompounds having co-surfactant action to form w/o microemulsions. Thesystem studied by the Authors consisted of mixtures of a fatty acidester, a 1:1 lecithin-co-surfactant and water in various ratios. Anefficiency scale of the compounds used as co-surfactants has beendefined: primary amines>alcohols>fatty acids. Furthermore, theefficiency proved to be related with the length of the alkyl chain ofthe alcohol and of the respective fatty acid; hencebutanol>pentanol>hexanol>pentanoic acid>hexanoic acid. On the basis ofthese experimental data, alcohols such as butanol and pentanol and, to aless extent, the corresponding fatty acids, appear to be the bestcandidates. The use of the described compounds allows to prepare stablew/o microemulsions, but does not ensure the tolerability of saidformulations, particularly for the depot use wherein the formulation andthe subcutaneous or intramuscular tissue remain in contact for days oreven weeks.

Moreover, literature reports that the co-surfactant to surfactant ratiois critical; the data reported by Aboofazeli et al. relate to a 1:1ratio between the two components. Atwood et al (Int.J.Pharmacy 84 R5(1992)) studied the behaviour of lecithin/water/fatty acid ester/butanolmixtures wherein the surfactant/co-surfactant ratio is increased fromabout 1.7 to 3. The Authors evidenced that the decrease of theco-surfactant amount in favor of lecithin dramatically restricts thearea in which the presence of a w/o microemulsion is observed, even whenusing a remarkably efficient co-surfactant such as butanol.

Surfactants are generally classified according to an empirical scale,known as hydrophile-lipophile balance (HLB) which assignes valuesranging from 1 to 40. As a rule, suitable surfactants for w/omicroemulsions have low HLB whereas those suitable for o/wmicroemulsions have high HLB. When the interfacial tension is <2×10⁻²dyn/cm, a stable microemulsion can form. The optional presence of aco-surfactant allows to increase the interfacial fluidity, as theco-surfactant molecules penetrate between the surfactant molecules,thereby producing a dishomogeneous surface film. Co-surfactants can alsodecrease the aqueous phase hydrophilia and hence the interfacial tensionbetween the two phases. In principle, the use of co-surfactants isadvantageous in that they allow to decrease the amount of necessarysurfactant while increasing the stability of the microemulsion; however,as already mentioned above, it is preferable to limit their use as theyhave potential topical toxicity, mainly in case of contact between thecarrier and the subcutaneous or intramuscular tissues for prolongedtimes.

The numerous advantages of the use of a microemulsion as a carrier foractive ingredients are known.

Microemulsions, under specific conditions of ratios between thecomponents, can form spontaneously without need for high power for thepreparation thereof; therefore their preparation on an industrial scalecan be easy. Said spontaneous microemulsions are thermodynamicallystable, homogeneous and transparent, so that they can be monitored byspectroscopical techniques. Microemulsions with mean diameters <100 nmcan be prepared, which can be cold sterilized by filtration trough 0.22micron membrane commercial filters. The microemulsions can allow toadminister poorly soluble or poorly stable drugs.

Furthermore said systems can undergo phase inversion when an excess ofthe dispersed phase is present, or as a consequence of a change oftemperature: this property can affect the bioavailability of the activeingredient with a mechanism that has not yet been clarified.

The w/o microemulsions can control the release of the active ingredientor protract its stability in physiologic fluids through protection fromthe action of hydrolytic enzymes. A number of reviews on microemulsionsexist, for example “Industrial application of microemulsions” MarcelDekker Ed. 1997, which in the chapter “Microemulsions in thePharmaceutical field: perspectives and applications” deals with theusefulness thereof in the pharmaceutical field, and “Handbook ofMicroemulsion Technology”, Ed. Kumar Mittal (1999) concerning thechemical-physical aspects.

The use of w/o microemulsions as vehicles to obtain a controlled releaseof active ingredients which are hydrophilic or are made hydrophilic bysuitable derivatization, is reported in the patent literature.

In particular, as for biodegradable molecules such as peptides, theparenteral administration of w/o microemulsions containing said activeingredients is reported in a study [M. R. Gasco et al., Int.J.Pharm.,62, 119 (1990)] wherein an LHRH hormone analog, formulated in amicroemulsion consisting of components considered biocompatible andcontaining 500 μg/ml of the active ingredient, administered by singleintramuscular injection at doses of 3 mg/Kg in adult male rats weighingabout 200 g, decreased testosterone plasma levels for a time up to about30 days after the injection; said levels were lower than those observedin a second group of mice treated with repeated injections (one a dayfor 28 days) at doses of 100 μg/Kg of active ingredient in buffersolution.

It should however be noted that the decrease in the testosteroneconcentration was not homogeneous in time during observation, and itbecame therapeutically effective only 8 days after the administration.

This delay in the therapeutical effect is not very advantageous,particularly in the treatment of prostate tumor which is known torequire testosterone for growing; therefore the faster the testosteronenormal production is stopped, the more effective the treatment.

The above mentioned paper shows the effectiveness of a w/o microemulsionconsisting of ethyl oleate (60.5%), water (10.1%), phosphatidyl choline(18.9%) and caproic acid (10.5%) for the sustained release of thepeptide. The surfactant (phosphatidylcholine) to co-surfactant (caproicacid) ratio is 1.8. The components of said microemulsion, individuallytaken, are considered biologically compatible by the Authors. No mentionis made of the biocompatibility of the microemulsion as a whole neitherof the condition of the subcutaneous tissue in contact with theformulation.

According to the teaching of said paper, a w/o microemulsion containingLeuprolide acetate as active ingredient with LHRH activity was prepared,consisting of ethyl oleate (66.9%), water (9.7%), phosphatidyl choline(19.4%) and a caproic acid/butyric acid 3/1 mixture (3.9%). According tothis paper, the mixture with butyric acid was used instead of caproicacid alone in order to minimize the surfactant to co-surfactant ratio,which is in this case 4.9 instead of 1.8. The in vivo test carried outby subcutaneous injection of the product in the rat has confirmed theeffectiveness but has also surprisingly shown alarming localulcerogenicity and persistent formation of subcutaneous granulomas. Thispharmacologically unacceptable result, notwithstanding the lower amountsof co-surfactants used, proved that the biocompatibility of the singlecomponents of the microemulsion was not sufficient to ensure thebiocompatibility of the mixture constituting the microemulsion, whenused for the parenteral administration.

Similarly, the mixtures disclosed and claimed in various patents, e.g.WO 94/08610, although providing stable microemulsions and possiblecontrolled releases of the active ingredient in time, do not teach tothose skilled in the art how to obtain the sustained release of anhydrophilic active ingredient while avoiding such side effects. Saidmicroemulsions usually consist, in fact, of water, an oily component, asurfactant, a co-surfactant, and optionally electrolytes, in variousratios. Neither biocompatibility of the microemulsion “in toto” isevaluated, nor the necessary ratios of mixture components to activeingredient are indicated, to obtain stability of both the activeingredient and the microemulsion as well as biocompatibility of theformulation.

On the other hand, said patents did not specifically consider theproblems concerning local tolerability connected with intramuscular(i.m.) and subcutaneous (s.c.) administrations, which are the mostsuitable and easy routes for the parenteral administration.

An alternative technology already used at the industrial level for thesustained release of peptides is that described and claimed in a numberof patents, inter alia U.S. Pat. No. 3,976,071, wherein such release isobtained by the use of bioerodible polymers in which the activeingredient is inbodyrated. Typical examples of bioerodible polymers arepolymers based on glycolic acid and lactic acid. The drawback of saidtechnology is that it is relatively expensive and troublesome comparedwith the above described microemulsions, furthermore it requires the useof organic, in particular chlorinated, solvents during the preparation,which involves problems in terms of environmental impact and safety ofthe pharmaceutical formulation.

On the other hand, said formulations advantageously cause not persistentgranulomas and do not induce local ulcerogenicity.

DISCLOSURE OF THE INVENTION

The present invention aims at providing sustained release pharmaceuticalcompositions in the form of stable w/o microemulsions, which are easy toprepare, can be sterilized, are free from remarkable systemic or topicalside effects, are suitable for parenterally administering, preferablyi.m. or s.c., active ingredients which are hydrophilic or are madehydrophilic through suitable derivatization, thereby obtaining aremarkable technical improvement compared with the known technique.

A procedure has been found, consisting in the clinical, post-mortem, andhistological examinations of the animals treated with the abovementioned microemulsions, suitable for thoroughly evaluating thebiocompatibility of any formulations for the sustained releaseadministration.

According to this procedure, a microemulsion is considered acceptable,according to what stated in literature (Protein Formulation andDelivery—F. J. McKelly 2000 pages 245–247), when any local swelling,more or less marked depending on the administered dose, is anywayreversible; on the other hand, a similar tissular response is alsoobserved for materials considered biodegradable, which response isapparently important in affecting the sustained release of the drug intime. A local intolerance in the form of persistent ulcerations isconversely considered unacceptable.

The microemulsions of the present invention consists for up to 20% of aninternal hydrophilic aqueous phase containing the active ingredient, for30 to 98% of a hydrophobic external phase and for up to 50% of asurfactant alone or in admixture with a co-surfactant. The microemulsionpreferably contains a percentage ≦35% of surfactant and co-surfactant,with a surfactant/co-surfactant ratio ≧2, and most preferably ≧3,5.

Other biologically compatible excipients which do not affect thestability of the microemulsion can also be present.

Suitable surfactants for the microemulsions of the invention areselected from natural or synthetic glycerophospholipids containingresidues of C₄–C₂₀ saturated or unsaturated carboxylic acids, having asphosphoester moiety a residue of choline, ethanolamine, serine,glycerol; cholesterol; C₁₂–C₂₀ fatty acid esters of sugars such assorbitol, galactose, glucose, saccharose; polyoxyethylene sorbitanC₁₂–C₂₀ fatty acid esters.

The optionally present co-surfactants are selected from C₈–C₂₀ fattyacids, C₂–C₁₄ polyhydroxyalkanes, particularly propylene glycol,hexanediol and glycerol, C₂–C₁₂ alcohols, esters of lactic acid with aC₂–C₈ alcohol residue.

The hydrophobic continuous phase is selected from the followingcompounds, alone or in mixture: esters of C₈–C₂₀ saturated orunsaturated carboxylic acids with a C₂–C₈ alcohol residue or mono, di-and triglycerids of C₈–C₂₀ fatty acids, or vegetable oils suitable forthe parenteral administration, such as soybean, peanut, sesame,cottonseed, sunflower oils.

The microemulsions of the invention are further characterized by pHranging from 4.5 to 7.5, preferably from 5 to 7, said pH, when notintrinsic to the composition of the microemulsion, being preferablyobtained by addition of a suitable amount of a natural amino acid to themicroemulsion without affecting its stability and the average size ofthe droplets.

The w/o microemulsions of the invention are particularly suitable ascarriers for peptides, in particular LHRH analogs such as Leuprolideacetate, Goserelin, Triptorelin, Nafarelin acetate, Histrelin,Cetrorelix or the corresponding acetates, or peptides such asSomatostatin or its analogs such as Octreotide and Lanreotide acetate.

Furthermore, the microemulsions of the invention are particularlysuitable as carriers for polysaccharides, in particular unfractionedheparin or low molecular weight heparins.

The microemulsions of the invention allow to prepare formulations withsustained release of hydrophilic active ingredients. Said sustainedrelease formulations, which are a further object of the invention,induce no local ulcerogenicity and produce non persistent granulomas,which are reabsorbed during the time in which the medicament iseffective. In the case of LHRH-type peptides, and in particularLeuprolide, Goserelin, Triptorelin, Nafarelin acetate, Histrelin,Cetrorelix and the corresponding acetates, sustained release for atleast 30 days can be obtained. In the case of Somatostatin, Octreotideand Lanreotide, sustained release for at least 8 days can be obtained.

The invention further relates to the use of a microemulsion according tothe invention comprising Leuprolide, Goserelin, Triptorelin, Nafarelinacetate, Histrelin, Cetrorelix or the corresponding acetates for thepreparation of a medicament for suppressing testosterone productionafter single administration for at least 30 days, where testosteronelevel already decreases 48 h after the administration.

The invention also relates to the use of the microemulsions containingOctreotide or its analogues for the preparation of a medicament forsuppressing growth hormone production for at least 8 days.

A further object of the present invention is the use of themicroemulsions containing unfractioned heparin or low molecular weightheparins for the preparation of sustained release medicaments uponsingle administration.

The following examples further illustrate the present invention.

EXAMPLE 1 Preparation of a w/o Microemulsion Containing LeuprolideAcetate

a) Preparation of the Aqueous Phase

350 mg of Leuprolide acetate are dissolved in 10 ml of water forinjections added with 200 mg of lysine.

b) Preparation of the Oily Phase

60 g of ethyl oleate, 25 g of soy lecithin (purity >95%) and 5 g ofcaprylic acid are mixed separately, in a suitable vessel thermostatizedat a temperature of 60–70° C., under stirring. The resulting clearhomogeneous solution is cooled at room temperature.

c) Preparation of the Microemulsion

The aqueous phase (solution a) is added to the oily phase (solution b)under stirring, to obtain an optically transparent, homogeneousmicroemulsion. A pH value of about 6 was evaluated based on the usedamounts of lysine and caprylic acid fraction soluble in the aqueousphase.

Said microemulsion is sterile filtered through a suitable 0.22 μmmembrane.

The Leuprolide acetate content of said microemulsion was evaluated byHPLC analysis in the following conditions:

Stationary phase: Vydac C18 5 μ column (250 × 4 mm) Mobile phase A:H₂0 + 0.1% TFA B: CH₃OH + 0.1% TFA Gradient: 20′ 100% A to 100% B Flow:0.8 ml/min Detector: UV 214 nm

The content in Leuprolide acetate is 3 mg/ml.

EXAMPLE 2 Preparation of a w/o Microemulsion Containing LeuprolideAcetate

The procedure of Example 1 is followed, but solubilizing 600 mg ofLeuprolide acetate in 10 ml of water.

EXAMPLE 3 Preparation of a w/o Microemulsion Containing LeuprolideAcetate

The procedure of Example 1 is followed, but without adding 200 mg oflysine. Calculated pH is about 3.

EXAMPLE 4 Preparation of a w/o Microemulsion Containing LeuprolideAcetate

The procedure of Example 1 is followed, but solubilizing 900 mg ofLeuprolide acetate in 10 ml of water.

EXAMPLE 5 Preparation of a w/o Microemulsion Containing LeuprolideAcetate

The procedure of Example 1 is followed, but changing the amounts ofsurfactant and co-surfactant to 15 g of soy lecithin and 3 g of caprylicacid, respectively. In this way, although keeping the ratio between thetwo components unchanged (5:1) the total amount of thesurfactant/co-surfactant mixture is changed from 30% to 18%.

EXAMPLE 6 Preparation of a w/o Microemulsion Containing Octreotide

The procedure of Example 1 is followed, but solubilizing 3 g ofOctreotide in 10 ml of water, instead of 350 mg of Leuprolide acetate in10 ml of water.

EXAMPLE 7 Preparation of a w/o Microemulsion Containing Heparin

The procedure of Example 1 is followed, but solubilizing in 10 ml ofwater 50 mg of unfractioned heparin in the form of calcium or sodiumsalt, instead of 350 mg of Leuprolide acetate.

EXAMPLE 8 Preparation of a w/o Microemulsion Containing LeuprolideAcetate

The procedure of Example 1 is followed, but changing thequali-quantitative composition of the oily phase: ethyl oleate (66.9%),phosphatidyl choline (19.4%) and a 3:1 caproic acid-butyric acid mixture(totally 3.9%).

EXAMPLE 9 Preparation of a w/o Microemulsion Containing LeuprolideAcetate

a) Preparation of the Aqueous Phase

60 mg of Leuprolide acetate are dissolved in 0.6 ml of water forinjections added with 8 mg of lysine.

b) Preparation of the Oily Phase

2.1 g of ethyl oleate, 215 mg of polyoxyethylene sorbitan monooleate and1 g of soy lecithin are mixed in a suitable vessel, heating at 50° C.under stirring. The resulting clear homogeneous mixture is cooled atroom temperature.

The aqueous solution is slowly added in portions to the oily mixtureunder stirring, to obtain an optically clear microemulsion containing1.5% of Leuprolide acetate.

EXAMPLE 10 Preparation of a w/o Microemulsion Containing LeuprolideAcetate

a) Preparation of the Aqueous Phase

60 mg of Leuprolide acetate are dissolved in 0.2 ml of water forinjections added with 4.1 mg of lysine.

b) Preparation of the Oily Phase

1.2 g of ethyl oleate, 0.5 g of polyoxyethylene sorbitan monooleate and0.1 g of caprylic acid are mixed in a suitable vessel, heating to 50° C.under stirring. The resulting clear homogeneous mixture is cooled atroom temperature.

The aqueous solution is slowly added in portions to the oily mixtureunder stirring, to obtain an optically clear microemulsion containing 3mg/ml of Leuprolide acetate.

EXAMPLE 11 Preparation of a w/o Microemulsion Containing OctreotideAcetate

a) Preparation of the Aqueous Phase

20 mg of Octreotide acetate are dissolved in 0.2 ml of water forinjections containing 0.2 g of propylene glycol and 4 mg of lysine.

b) Preparation of the Oily Phase

0.98 g of ethyl oleate, 0.5 g of soy lecithin and 0.1 g of caprylic acidare mixed in a suitable vessel, heating to 50° C. under stirring. Theresulting clear homogeneous mixture is cooled at room temperature.

The aqueous solution is slowly added in portions to the oily mixtureunder stirring. The resulting optically clear microemulsion contains 10mg/ml of Octreotide acetate.

EXAMPLE 12 Preparation of a w/o Microemulsion Containing OctreotideAcetate

a) Preparation of the Aqueous Phase

10 mg of Octreotide acetate are dissolved in 0.1 ml of water forinjections added with 2 mg of lysine.

b) Preparation of the Oily Phase

0.59 g of ethyl oleate, 0.25 g of soy lecithin and 0.05 g of caprylicacid are mixed in a suitable vessel, heating to 50° C. under stirring.The resulting clear homogeneous mixture is cooled at room temperature.

The aqueous solution is slowly added in portions to the oily mixtureunder stirring. The resulting optically clear microemulsion contains 1%of Octreotide acetate.

EXAMPLE 13 Preparation of a w/o Microemulsion Containing OctreotideAcetate

0.59 g of ethyl oleate, 0.25 g of soy lecithin and 0.05 g of caprylicacid are mixed in a suitable vessel, heating to 50° C. under stirring.The resulting clear homogeneous mixture is cooled at room temperature.0.1 g of water for injections containing 2 mg of lysine, are graduallyadded under stirring to the oily solution. The resulting optically clearmicroemulsion is added under stirring with 10 mg of Octreotide acetate.The active ingredient is inbodyrated in said microemulsion within a fewseconds. The microemulsion is filtered through a 0.22 mcm polysulfonefilter. The resulting optically clear microemulsion, analyzed by HPLC,shows a content in Octreotide acetate of 6.35 mg/ml.

EXAMPLE 14 Preparation of a w/o Microemulsion Containing Myoglobin

a) Preparation of the Aqueous Phase

5.5 mg of Myoglobin are dissolved in 1.5 ml of water for injectionsadded with 10 mg of lysine.

b) Preparation of the Oily Phase

2 g of ethyl oleate, 1.2 g of soy lecithin and 0.25 g of caprylic acidare mixed, in a suitable vessel, heating to 50° C. The resulting clearhomogeneous mixture is cooled at room temperature.

The aqueous solution is slowly added in portions to the oily mixtureunder stirring. The resulting optically clear microemulsion contains 1.3mg/ml of Myoglobin.

EXAMPLE 15 In vivo Evaluation of the Effectiveness of the MicroemulsionContaining Leuprolide Acetate Prepared as Described in Examples 1 and 2.

Three groups of Sprague Dawley male rats (10 per each group) were housedfor 5 days with water and food ad libitum before treatment.

Two microemulsion formulations prepared as described in Examples 1 and2, with different concentrations of Leuprolide acetate, namely 3 mg/ml(Example 1) and 6 mg/ml (Example 2) were administered in a single dose,each in a group of rats, at the dose of active corresponding to 0.750mg/kg.

Control animals (3 per group) received saline solution.

Testosterone plasma levels after taking the blood samples were evaluatedat days 1, 2, 3, 4, 7, 14, 21, 28, 42 and 56. Blood samples werecentrifuged and serum testosterone was measured by EIA kit.

Serum testosterone levels were evaluated until 60 days afteradministration.

The results are described in FIG. 1.

The following Table 1 summarizes the data concerning organs weight inthe treated animals.

TABLE 1 Effect of the microemulsions prepared as described in Examples 1and 2 on body and reproductive organs weight in rats. Body andreproductive organs weight (g) after 28 and 56 days 28 days 56 daysSeminal Seminal Microemulsion Body Testes Prostate vesicles Body TestesProstate vesicles Salina 570.0 ± 13.2 3.73 ± 0.09 0.72 ± 0.07 1.95 ±0.010 631.2 ± 10.1 3.54 ± 0.05 0.80 ± 0.05 1.96 ± 0.26 Example 1 546.6 ±8.8 1.84 ± 0.11* 0.33 ± 0.03* 0.49 ± 0.08* 600.0 ± 16.4 2.39 ± 0.17*0.50 ± 0.03^(#) 1.39 ± 0.14 Example 2 532.8 ± 4.6 1.68 ± 0.14* 0.22 ±0.03* 0.25 ± 0.03* 626.6 ± 25.5  2.5 ± 0.2^(⋄) 0.51 ± 0.08^(#) 1.43 ±0.22 Data expressed as mean ± standard error of the group (5 animals) *p< 0.001; ^(⋄)p < 0.01; ^(#)p < 0.05 vs. saline (ANOVA test andBonferroni † test for multiple comparisons).

The formulations of the present invention, independently on theconcentration of the active in the microemulsion, are free fromsubstantial systemic or local side effects and provide the sustainedrelease of the active ingredient for a duration of at least 30 days andtherapeutic efficacy already 48 h after the injection.

The efficacy is comparable to that obtainable when using the commercialdepot formulation “Enantone” based on bioerodible polymers.

EXAMPLE 16 Evaluation of the Subcutaneous Tolerability of theMicroemulsions

The test for the evaluation of the subcutaneous tolerability waseffected by injecting groups of at least 9 rats with a singleadministration of the microemulsions reported in the following Table 2.

Clinical, post-mortem and histologic examinations were carried out 48 h,7 days and 14 days after the administration.

TABLE 2 Presence of swelling Presence of (Mean score *) ulcerationsAmount 14 14 Compound (mg) 48 h 7 days days 48 h 7 days daysMicroemulsion 125 μl 2 2 1 No No No A⁽¹⁾ Microemulsion 500 μl 3 3 2.5 NoNo No A⁽¹⁾ Microemulsion 125 μl 3 3 3 Yes Yes Yes B⁽²⁾ ⁽¹⁾Microemulsionprepared as described in Example 1. ⁽²⁾Microemulsion prepared asdescribed in Example 8. * disclosure of the score: 1 slight effect; 2moderate; 3 severe.

This test considers biologically compatible the formulations whichinduce no persistent local ulcerations, whereas the presence of swellingis a function of the amount of product injected and of the rate ofelimination of the ethyl oleate from subcutaneous tissues, as describedby Howard et al. in Int. J. Pharm. 16 (1983) 31–39.

EXAMPLE 17 In vivo Evaluation of the Effectiveness of the MicroemulsionContaining Octreotide Acetate

A dose corresponding to 6 mg/rat of microemulsion containing Octreotideprepared as described in Example 11 was administered to 6 male ratsweighing about 175–200 g. Plasma samples were taken before theadministration of the compound and 0.5 hours, 24 hours, 4, 6 and 8 daysafter treatment.

Octreotide was extracted from plasma and analyzed with LC-MS-MSapparatus vs. a calibration curve. Plasma levels are reported in thefollowing table 3:

TABLE 3 Time of sampling 0.5 h 24 h 4 days 6 days 8 days Octreotide 53940.2 62.5 4.5 7.6 (ng/ml)Significant Octreotide plasma levels were found until 8 days aftertreatment.

EXAMPLE 18 Evaluation of the Dose/Efficacy Ratio of the MicroemulsionContaining Leuprolide Acetate

The formulation containing Leuprolide acetate, prepared as described inExample 2, was administered to rats, in a single dose of 2.25 mg/kg(Example). Control animals received a corresponding dose of salinesolution (Control).

Testosterone plasma levels on blood samples were evaluated at days 0, 1,2, 3, 5, 7, 14, 21, 28, 42 and 56. Blood samples were centrifuged andserum testosterone was measured by an Elisa kit.

Results are shown in FIG. 2.

The data concerning organs weights in treated animals are summarized inthe following Table 4.

Table 4: Effect of a Leuprolide acetate microemulsion or of a salinesolution on the body and reproductive organs weight in rats (Example andControl).

Body and reproductive organs weight (g) on 56 day Body Testes ProstateSeminal vesicles Control 645 ± 34 3.73 ± 0.16 0.82 ± 0.04 1.96 ± 0.091.94 ± 0.42 0.35 ± 0.14 0.55 ± 0.16 Example 608 ± 35 (p < 0.01) (p <0.5) (p < 0.05)

Reduction of both testosterone plasma levels and reproductive organsweight is clearly evidenced 56 days after the administration of themicroemulsion.

1. A water-in-oil (w/o) microemulsion comprising: a) up to 20% of aninternal hydrophilic aqueous phase containing a therapeutically activehydrophilic compound; b) 30 to 98% of a hydrophobic external phaseselected from esters of C₈–C₂₀ saturated or unsaturated carboxylic acidswith a C₂–C₈ branched or linear alcohol residue or mono, di- andtriglycerides of C₈–C₂₀ fatty acids; c) up to 50% of a surfactant inadmixture with a co-surfactant, said surfactant being selected fromnatural or synthetic glycerophospholipids containing residues of C₄–C₂₀saturated or unsaturated carboxylic acids having as phosphoester moietya residue of choline, ethanolamine, serine, glycerol, and saidco-surfactant being selected from C₈–C₂₀ fatty acids or C₂–C₁₂ alcohols,and wherein the surfactant/co-surfactant ratio is ≧2; wherein theaqueous phase has a pH between 4.5 and 7.5.
 2. The microemulsion ofclaim 1, which induces no local ulcerogencity, but which may producenon-persistent granulomas.
 3. The microemulsion of claim 1, wherein thetherapeutically active compound comprises a peptide, protein,oligosaccharide or polysaccharide.
 4. The microemulsion of claim 1,wherein the therapeutically active compound is a peptide.
 5. Themicroemulsion of claim 1, wherein the therapeutically active compound isan LHRH hormone peptide analog.
 6. The microemulsion of claim 1, whereinthe therapeutically active compound is Leuprolide acetate, Goserelin,Triptorelin, Nafarelin, Histrelin, Cetrorelix or the correspondingacetates.
 7. The microemulsion of claim 1, which provides sustainedrelease of the therapeutically active compound for at least 30 days. 8.The microemulsion of claim 1, wherein the therapeutically activecompound is Somatostatin, Octreotide or Lanreotide.
 9. The microemulsionof claim 1, which provides sustained release of the therapeuticallyactive compound for at least 8 days.
 10. The microemulsion of claim 1,wherein the therapeutically active compound is an oligosaccharide or apolysaccharide.
 11. The microemulsion of claim 1, wherein thetherapeutically active compound is unfractioned heparin or one or morelow molecular weight heparins.
 12. The microemulsion of claim 1, whereinthe therapeutically active compound has been made hydrophilic by beingderivatized.
 13. The microemulsion of claim 1, wherein the surfactant isselected from the group consisting of: a) natural or syntheticglycerophospholipids containing residues of at least one C₄–C₂₀saturated or unsaturated carboxylic acids, having as phosphoester moietya residue of choline, ethanolamine, serine, or glycerol; b) cholesterol;c) sugars of C₁₂–C₂₀ fatty acid esters; and d) polyoxyethylene sorbitanC₁₂–C₂₀ fatty acid esters.
 14. The microemulsion of claim 1, wherein theco-surfactant is selected from the group consisting of C₈–C₂₀ fattyacids; C₂–C₁₄ polyhydroxyalkanes; C₂–C₁₂ alcohols; and esters of lacticacid with a C₂–C₈ alcohol residue.
 15. The microemulsion of claim 1wherein the hydrophobic continuous phase comprises one or more of thefollowing compounds: an ester of a C₈–C₂₀ saturated or unsaturatedcarboxylic acid with a C₂–C₈ alcohol residue, a mono, di- ortriglyceride of a C₈–C₂₀ fatty acid, or a vegetable oil.
 16. Themicroemulsion of claim 1, wherein the aqueous phase has a pH between 5and
 7. 17. The microemulsion of claim 1, further comprising one or morenatural amino acids to adjust the pH to 4.5 to 7.5.
 18. A method forpreparing a medicament comprising: admixing the microemulsion of claim 1with Leuprolide, Goserelin, Triptorelin, Nafarelin acetate, Histrelin,Cetrorelix or the corresponding acetates or other LHRH analogs, andformulating said medicament to suppress testosterone production aftersingle administration for at least 30 days, and to decrease thetestosterone level 48 h after the administration.
 19. A method forpreparing a medicament comprising: admixing the microemulsion of claim 1with Octreotide or its analogues, and formulating said medicament tosuppress growth hormone production for at least 8 days.
 20. A method forpreparing a medicament comprising: admixing the microemulsion of claim 1with unfractioned heparin or one or more low molecular weight heparins,wherein said medicament is formulated for sustained release after asingle administration.
 21. A water-in-oil (w/o) microemulsion consistingof: a) up to 20% of an internal hydrophilic aqueous phase containing atherapeutically active hydrophilic compound which has a pH between 4.5and 7.5; b) 30 to 98% of a hydrophobic external phase selected fromesters of C₈ 14 C₂₀ saturated or unsaturated carboxylic acids with aC₂–C₈ branched or linear alcohol residue or mono, di- and triglyceridesof C₈–C₂₀ fatty acids; c) up to 50% of a surfactant in admixture with aco-surfactant, said surfactant being selected from natural or syntheticglycerophospholipids containing residues of C₄–C₂₀ saturated orunsaturated carboxylic acids having as phosphoester moiety a residue ofcholine, ethanolamine, serine, glycerol, and said co-surfactant beingselected from C₈–C₂₀ fatty acids or C₂–C₁₂ alcohols, and wherein thesurfactant/co-surfactant ratio is ≧2.
 22. The water-in-oil (w/o)microemulsion of claim 21, which contains ≦35% of asurfactant/cosurfactant mixture having a ratio of surfactant tocosurfactant of ≧3.5.